Electronic particle counting apparatus



Jan. 17, 1956 w. s.. PIKE 2,731,202

ELECTRONIC PARTICLE COUNTING APPARATUS Filed April 5. 1951 United States Patent ELECTRONIC PARTICLE COUNTING APPARATUS Winthrop Seeley Pike, Princeton, NL J., assigner to Radio Corporation of America, a corporation of Delaware Application April 3, 1951, Serial No. 219,006

6 claims. (Cl. 23S- 92) This invention relates to improvements inapparatus for counting uniformly shaped discrete particles spread out` in a eld of view. By particles is meant notonly physical entities having three dimensions, but any discrete areas or spots having an appearance contrasting with that ofthe general background. While not limited thereto, the present invention finds particular application in systems for determining blood cell count, and it will be described with special reference thereto.

@ne of the most frequent laboratory tests performed in hospitals is the red blood cell count, for the concentration ot` such cells in the blood is very greatly changed in certain forms of illness. The usual technique for performing this test is exacting, laborious and' time consuming and may, in the case of large hospitals, requirev` the services of a considerable number of skilled technicians'. The procedure is as follows: A sample of the patients blood is accurately diluted to one part of blood in two hundred parts of diluting solution. The diluting solution used in making a red cell count may be chosen to destroy the white cells, thus leavingy only red cell's visible in the counting microscope. After agitation to insure thorough mixing, a minute quantity of the diluted blood is placed in a ruled counting chamber of known volume, under a microscope. The rulings define the volurne within which cells are to be counted'. VThetechnician then observes thel blood within the standard volume of the chamber by means ofthe microscope and manually counts the cells within that volume, multiplying the result by the appropriate factor to arrive at the number of cells per cubic millimeter in the original sample. In normal blood this ligure isV about tive million andthe dilution is such that the technician must count several hundred cells. It will be readily appreciated that the process is tedious, fatiguing 'andhasl many opportunities for human error. In practice, a skilled technician can make only about six counts per hour withV reasonable accuracy.

Machine counting offers an appreciable saving 'in time, and therefore money, as well as improved accuracy. Tests with an apparatus of the type disclosed herein indicate that tive per cent accuracy may be expected. An accuracy of ten per cent is considered typical for red blood cell counts performed in the conventional manner.

It is a general object of the present invention to provide an improved apparatus for automatically counting uniformly shaped particles, and one that is particularly applicable to the problem of' blood cell counting.

A more specific object ofthe invention is to provide a particle counting apparatus wherein errors due to differcnces in particle size are minimized.

In accordance with the invention, the foregoing and other related objects and advantages are attained in a system wherein a eld of view in which particles appear is scanned with a beam of energy. After the beam has impinged on the viewing field, changes in beam intensity (due to particle interceptions of the beam) are converted 1o electrical pulses. These pulses are counted.' The rice average pulse width also isv determined as a measure of average particle: size. This pulse width information then is supplied to the pulse counting section of the system as a correction factor to minimizev errors in particle count resulting from variations in` particle size.

A more complete understanding of'` the invention can be had by reference t'o the following description of. illustrative embodiments thereof, when considered in connection with the accompanying. drawing, wherein:

Figure l is a schematic diagram illustrating generally a particle counting system arrangedl in accordance with the invention,

Figure 2 shows an` alternative form of particle-representative-pulse generator suitable for use in a system of the type shown in Figures ly and 3, and

Figure 3 is aI schematic diagram showing circuit details of an apparatus arrangedk in accordance with the invention.

In the system shownV in Fig. l, the fieldy of. view contaiuingparticles to bevcountedf is.` assumed' to comprise an unruled counting chamber, 10 containing a blood sample, prepared as previously explained and' arranged' ony the stage llt of a microscope 12.

` A televisionk camera 14 is mounted with its viewing lens` 16 facing-against the eyepiece 1'8 ofV the-*microscope 1-2. As usual, the electron beam in the camera will raster scan the' field of: view presented thereto. The termraster scanning, as used herein and in the ap.- pended claims, is intended to mean repeated movement of'a beam of energy along a multiline pattern, with each line. in the patterny being parallel to andA slightly dis.- placed from the preceding line. Such al scanning: pattern is well knownin the television art and'y allied ields'.

As the electron beam inthe camera 14 scans the field ofview presented through the' microscope optical system, the usual video television signal will be generated This video signal preferably'is supplied to a monitoring television receiver 20, to provide on the viewing screen 2.2v thereof a picture-'of the-field' of view being examined'. This, of course, facilitates optimum adjustment of the microscope and camera controls. It has been-found that in a televisionv pulse generating systemof the type being described, optimum results are obtained with so-calleddark' eld illumination.

As is well known, thesignals generated in the television camera 14 will include-pulses representative of the particles appearing in the field of View of the camera. These pulses are applied to separate channels in the system of: Fig. 1. ln the first channel, the pulses are counted in a pulse counting circuit 24 to provide ona meter 26' an indication of thenumber of pulses counted. For example, the metering circuit may comprise a socalled integrating count rate meter, a specific example of which isV given hereinafter.

If the size of thel particles being counted is constant, then the pulse count indicated by the meter 26 will be quite accurately proportional to the number of'pa-rticles in the field of View. in practice, however, this isusually not the case. In the case of blood cell counting', the meanl diameter of red blood' cells shows somey variation from person to person. Also, some diseasesresult in changes in cell diameter. These diameter variations will tend to introduce an error, as' Will now. be explained.

Each particle in the'ield'will be scanned by more than oneline of the television raster and zmay, thus, be counted several times. Obviously, larger cells will be scanned moreV timesthan small cells'. Therefore, in an expres-` sion relating pulse count to the number of Acells in'the eld, the cell diameter` would appear as-a variable which should be eliminated. Since the cellsare' round, and since very. few linesof the raster will pass enact-ly.throughA the cell center in cutting across cells, most of the pulse widths will be shorter than the cell diameter. However, probability calculations have indicated that the most probable diameter diiers from the real diameter by a small and, more important, practically constant amount. This etect would then be small if the shape of the cell remains constant. In practice, the error so produced has proven negligible.

Since the pulse widths produced will be directly proportional to the cell diameters, it is possible to determine the unknown diameter term in the expression relating pulse count to particle number. Therefore, the accuracy of the counting process will hinge on having means for measuring the length of the video pulses, and for correlating this pulse width information with the pulse count information in such manner as to correct for the variable particle size factor.

To this end, and in accordance with the invention, the second channel to which the pulses are applied in the apparatus of Fig. l comprises a pulse width detecting circuit 28. This circuit may take any one of a number of dilerent forms, one specific example being given hereinafter. In general, the circuit 28 should comprise some type of indicator 30, and a means for adjusting the pulse width to which the circuit will be most sensitive. In other words, the pulse width sensitivity control 32 would be adjusted to obtain a maximum reading on the meter 30. This would indicate that the sensitivity control has been set so that the circuit 28 is responding to the maximum number of pulses, or, conversely that the pulse width sensitivity control 32 has been adjusted to correspond with the average width of the video pulses. Further in accordance with the invention, this pulse width sensitivity control 32 can be ganged to a calibration control 34 for the meter 26. Thus, as the pulse width sensitivity control is adjusted to respond to the maximum number of pulses, the pulse counting circuit sensitivity will be furnished with a correction factor which will minimize the error due to variations in particle size. Without this correction, a total of, say, 5,000 particles of one given average diameter might produce the same reading on the meter 26 as a total of, say, 4,500 cells of dilferent average diameter. The correction factor introduced has the effect of making the pulse count meter 26 indicate the number of pulses that would be produced if all the particles had a diameter-equal to their average diameter.

It will be understood that the present invention is not limited to the use of a pulse generator-scanner of the specific type shown in Fig. 1. For example, a photomicrograph or electron micrograph transparency may be made of the area containing the particles to be studied. The beamY of energy for scanning the area may be a focussed beam of light, scanned mechanically, or a focussed beam of light generated by a stream of electrons striking the fluorescent surface coating of a cathode ray kinescope tube. An arrangement of this latter type is shown in Fig. 2, and will be discussed hereinafter. The field of view may be either translucent or opaque to the beam of energy, and the residual energy of the beam may be detected either in a transmitted ray or a reflected ray. Where scanning" is referred to it is, therefore, meant to encompass any one of these equivalents. Further, the particles being counted may appear either as portions of the field of view relatively more opaque to the energizing beam of energy or relatively more transparent thereto. Where the entire eld is opaque, the particles may simply have a reflective property different from that of the general background.

The pulse generator-scanner system shown in Fig. 2, comprises a kinescope tube 40, on the uorescent screen of which is produced a raster used as a ying spot light source. A lens system 42 is provided to image the raster on a transparency 44, which comprises a eld of view with particles therein to be counted. Another lens systern 46 may be used to focus the light transmitted through the transparency during the scanning process on a photoelectric cell 48. The photoelectric cell is connected to a voltage source 50 and load impedance 52 in the usual manner, to provide across the load impedance 52 pulses representative of changes in the light reaching the photocell 4S.

Turning now to a consideration of a more specic embodiment of the invention, the circuit shown in Fig. 3 includes a pulse generator-scanner 60 such as the television camera 14 of Fig. l or the scanning system shown in Fig. 2. Pulses generated by scanning of the viewing field 10 may be ampliiied and clipped in a conventional amplifier and clipper circuit 62 to provide pulses, A', of relatively uniform amplitude. Pulses from the ampliier and clipper 62 may be supplied to a differentiator circuit 64, the output of which will comprise a positive pip, B, corresponding to the leading edge of each particle-representing pulse, and a negative pip, C, corresponding to the pulse trailing edge. Following the differentiator, a phase inverter amplifier 65 is provided. At the amplifier-inverter output, the positive pips (B) will appear as negative pips, B1, and the negative pips (C) will appear as positive pips C1. The reason for this phase inversion will be brought out hereinafter.

From the output of the amplifier-inverter 65, two circuit paths are provided. One comprises a connection from the amplifier 65 to the control grid 68 of a pentode tube 66. The anode 70 of the tube 66 is connected through a meter 72 to an operating voltage source (not shown), designated B+. The tube cathode 74 is connected to an adjustable biasing network 76 of conventional form. A pair of resistors 7S, 80 connected in series to the voltage source B+ provides a voltage divider from which adjustable bias voltage can be applied to the tube cathode 74.

The adjustable resistor 80 normally is set so that a cut-olf bias will be applied to the tube 66. Therefore, the negative pips corresponding to the leading edges of the original pulses will produce no effect on the tube 66, while the positive pulses may produce plate current flow, depending on the condition of the tube suppressor grid 90, as is explained hereinafter.

The differentiated and inverted pulses also are applied through a coupling diode 82 and an inverter-amplifier 84 to a variable delay line 86. For example, the delay line 86 may be of the type described and claimed in the copending application of H. Kihn, Serial No. 176,324, filed July 28, l950 and assigned to the assignee of the present invention now Patent No. 2,619,537 issued November 25, 1952. A switch 88 is provided for connecting the delay line 36 to the suppressor grid 90 of the tube 66. With the switch 8S in this position, the suppressor grid 90 is grounded through a resistor 93. Due to the positive cathode voltage, the suppressor grid effectively will be receiving a cut-off bias voltage.

To determine the average diameter of the particles being counted, the switch S8 is placed in the position shown to connect the suppressor grid 90 to the adjustable delay line 86. Also, a switch 92, linked to the switch 88, is opened to remove an adjustable shunt 94 from the circuit of the meter 72. As to any pulse for which the delay of the variable delay line 86 is the same as the pulse width, positive pulses will arrive simultaneously at the control grid 63 and the suppressor grid 90 of the tube 66. This pulse coincidence will allow tube current to ow through the meter 72. rTherefore, it can be seen that the adjustment of the delay line 86 which provides maximum reading on the meter '72 will be an adjustment corresponding to the maximum number of pulses of that width, and hence, a measure of the average pulse width. At this point, it should be noted that the adjustable shunt resistor 94 'is mechanically linked to the delay line 36.

After this preliminary adjustment has been made, the circuit from the delay line 36 to the suppressor grid 90 is opened by actuating the switch 88. This will connect the suppressor grid 90 to the tube cathode 74, removing the cut-oli bias from the suppressor grid. Simultaneously, the switch 92 will shunt the resistor 94 across the meter 72. All positive pulses arriving at the tube control grid 68 now will cause plate current flow which will be integrated by the meter 72. By virtue of the mechanical connection between the meter-adjust resistor 94 and the adjustable delay line 86, adjustment of the latter automatically will select the value of shunt appropriate for a particular length of pulse. This arrangement automatically eliminates the variable diameter term from the expression relating pulse count, pulse width, and particle number, thereby minimizing the error due to particle size variation.

Tests made with an instrument such as that shown in Fig. 3 have indicated that an accuracy of plus or minus live per cent is attainable in blood cell counting. Furthermore, the instrument shown can be provided with means to indicate the average diameter of red blood cells, as, for example, a calibrated control 98 for the delay line 86. This is a quantity which may well have somc clinical value, although it has rarely been used heretofore because of the ditliculty of determining it.

What is claimed is:

l. In a particle counting apparatus of the type wherein a field of view, in which particles appear against a background contrasting in appearance with said particles, is scanned with a beam of energy, and wherein detecting means determine the number of times said particles intercept said scanning beam, the combination of means responsive to interception of said beam by said particles for determining the average diameter of said particles, and ganged, adjustable means coupling said detecting means and said diameter determining means to determine how many particles of diameter equal to said average is represented by said detecting means.

2. In a particle counting apparatus of the type wherein a eld of view, in which particles appear against a background contrasting in appearance with said particles, is scanned with a beam of energy, and wherein interceptions of said beam by said particles are detected by detecting means to determine the number of said interceptions, the combination of means responsive to said detecting means for determining the average diameter of said particles, and ganged, adjustable means coupled to said diameter determining means and to said detecting means for determining instantaneously the number of times said beam would be intercepted by said particles if all of said particles were oi diameter equal to said average diameter.

3. Apparatus for counting the number of particles appearing in a eld of view against a background contrasting in appearance with said particles, said apparatus comprising means for raster scanning said field of View with a beam of energy, a detector for detecting changes in the energy of said beam produced by interception of said beam by said particles, a rst circuit coupled to said detector for generating a first voltage impulse upon interception of said beam by each said particle and a second voltage impulse upon termination of each said interception, a second circuit coupled to said first circuit and conductively responsive to predeterminedly spaced pulses from said first circuit, said second circuit including means to vary said second circuit to adjust the pulse spacing to which said second circuit will respond, measuring means coupled to said second circuit to measure the integrated value of current flow therethrough, counting means coupled to said first circuit to count said voltage impulses, said counting means including an adjustable element for varying the response of said counting means to said voltage impulses, and a coupling between said adjusting means in said second circuit and said adjustable element to provide a proportional variation of said adjustable element upon adjustment of said adjusting means.

4. Apparatus for counting the number of particles appearing in a eld of view against a background contrasting in appearance with said particles, said apparatus comprising means for raster scanning said eld of view with a beam of energy, a detector for detecting changes in the energy of said beam produced by interception of said beam by said particles, a rst circuit coupled to said detector for generating a first voltage impulse upon interception of said beam by each said particle and a second voltage impulse upon termination of each said interception, a second circuit coupled to said lirst circuit and conductively responsive to predeterminedly spaced pulses from said first circuit, said second circuit including means to vary said second circuit to adjust the pulse spacing to which said second circuit'will respond, measuring means coupled to said second circuit to measure the integrated value of current flow therethrough, counting means coupled to said first circuit to count said voltage impulses, said counting means including a count indicator, and means coupling said count indicator to said adjusting means in said second circuit to vary the indications provided by said count indicator in accordance with changes in the adjustment of said second circuit adjusting means.

5. Apparatus for counting the number of particles appearing in a tield of View against a background contrasting in appearance with said particles, said apparatus comprising means for raster scanning said eld of view with a beam of energy, a detector for detecting changes in the energy of said beam produced by interception of said beam by said particles, a first circuit coupled to said detector for generating a rst voltage impulse upon interception of said beam by each said particle and a second voltage impulse upon termination of each said interception, a multigrid vacuum tube, a connection from one of said tube grids to said first circuit, a phase inverter, a pulse-delay device, a circuit including a rst switch for connecting said first circuit to another of said grids through said phase inverter and said delay device, a meter in circuit with said tube for indicating the integrated value of current flow therethrough, a variable resistor, means coupled to said resistor and to said delay device to vary the resistance of said resistor and the delay effect of said delay device proportionally, and a second switch mechanically coupled to said rst switch and arranged to connect said resistor in shunt with said meter upon opening of said rst switch.

6. Apparatus for counting substantially circular discrete particles appearing in a field of View against a background contrasting in appearance with said particles, said apparatus comprising means for scanning said eld with a beam of energy, means for detecting energy changes in said beam after it has impinged on said field, counting means responsive to detection of said changes by said detecting means for counting the number of said changes, means responsive to detection of said changes to determine the average diameter of said particles, and adjustable means to couple said counting means to said diameter determining means to obtain instantaneously an indication of the number of particles in said eld.

References Cited in the le of this patent UNITED STATES PATENTS 2,369,577 Kielland Feb. 13, 1945 2,412,467 Morton Dec. 10, 1946 2,415,190 Rajchman Feb. 4, 1947 2,415,191 Rajchman Feb. 4, 1947 2,419,914 Pamphilon Apr. 29, 1947 2,479,802 Young Aug. 23, 1949 2,480,312 Wolf Aug. 30, 1949 2,494,441 Hillyer Jan. 10, 1950 2,580,498 vAckerlind Ian. 1, 1952 2,584,052 Sandorf Ian. 29, 1952 2,610,541 Rowland Sept. 16, 1952 OTHER REFERENCES Popular Science (page May 1949. Proc. of the IRE, vol. 37, #5, Electronic Classifying Cataloguing and Counting Systems, by Parsons (pp. 564-568), May 1949. 

